Millions of Americans are affected by permanent hearing deficits and balance dysfunctions that result from losses of sensory hair cells. In mammalian ears, when hair cells die they are not effectively replaced, but the case is quite different in non- mammalian vertebrates. In those species hair cell loss leads to cell proliferation and the subsequent differentiation of new replacement hair cells, which then become innervated. These regenerative events lead to structural healing of damaged ears and can restore hearing and balance a few weeks after non-mammalian vertebrates have experienced damage that would result in permanent deficits for humans. Research has shown that human and rodent ear tissues can activate important elements of the biological machinery that underlies regeneration, but the regenerative responses in mammalian ears are normally limited. The research proposed seeks knowledge that may provide the means to eventually overcome those limits. This request for renewal of a project in its 24th year proposes to continue investigations that focus on identifying and understanding the signaling mechanisms that control the production of cells and the processes that lead to their specialization as sensory hair cells in embryonic and postembryonic ears. The mechanisms that limit self-repair in the ears of mammals will be investigated and treatments that may overcome those limits will be tested. The information sought may identify targets for the development of therapeutic approaches to stimulate self-repair in the ears of mammals. In addition, this project will seek to scale up the in vitro production of hair cells from lines of passaged cells. The availability of specialized cells that are produced in vitro has provided the basis for many examples of significant gains in cancer cell biology, neurology, and other fields. These cell lines and the knowledge gained in their further development and utilization holds the potential for contributing to improved treatments for diseases of the ear. The goals of this research directly pertain to possible recovery from sensorineural hearing loss and balance dysfunctions that contribute to prevalent communication disorders and to falls by elderly individuals. They also are likely to lead to improved understanding of the development of normal and abnormal auditory and vestibular function in human ears. With the proposed investigations, we seek to contribute to better understanding of the cellular lineages and the cellular mechanisms that contribute to the development, regenerative replacement, and in vitro production of inner ear hair cells. Hair cell loss is the leading cause of permanent hearing impairment as well as a contributing cause of balance impairments that can lead to falls in older individuals. The knowledge we seek holds the potential to ultimately contribute to the development of treatments that may lead to partial or complete recovery from forms of hearing or balance impairment that are now permanent.